Neurons are terminally postmitotic cells that utilize their microtubules for the elaboration of an elongated cellular process termed the axon. Microtubules form a continuous array from the cell body of the neuron into the most distal region of the axon termed the growth cone. Individual microtubules stop and start within the array and assume a variety of different lengths. Contemporary imaging studies have shown that only the shortest microtubules are in rapid transit down the axon, while the longer microtubules are essentially stationary. The short microtubules move in both directions, with about twice as many moving anterogradely as retrogradely. The motor proteins that transport axonal microtubules are not well understood. This grant application proposes to test a model called "cut and run," in which the short and long microtubules are subjected to the same motor-driven forces. The same motor-driven forces that act to rapidly transport the short microtubules serve to functionally integrate the longer microtubules with the substrate along which the shorter microtubules would be transported. Specifically, these substrates are bundles of actin filaments or other long microtubules. According to the model, the forces on the longer microtubules are crucial for offsetting the tendency of the axon to retract, and for enabling microtubules to actively participate in growth cone turning. The first specific aim seeks to identify the specific molecular motor proteins that transport short microtubules bi-directionally in the axon, and to ascertain the effects of these motors on the vitality of axonal growth and/or retraction. The second aim seeks to determine the mechanism by which forces generated by these same motors regulate changes in the distribution of microtubules during growth cone turning. The candidate motors are cytoplasmic dynein and three kinesins (kinesin-5, kinesin-12, and kinesin-14a) that are known to generate forces between microtubules in the mitotic spindle. The proposed experiments involve depletion of the motors by RNA interference as well as other methods for inhibiting the functions of the motors in a more acute and/or localized fashion. Microtubule behaviors will be assessed by live-cell imaging, and growth cone guidance will be evaluated by a number of different assays involving substrate, chemical, and physical cues. These studies will reveal fundamental mechanisms of neuronal development, and will also provide important clues for how axonal regeneration can be clinically augmented after injury.